Dissecting the variability of molecular phenotypes in population cell lines and single cells

In this thesis, the regulation of gene expression is analyzed in three different aspects. At first by understanding how the genetic variation has a downstream effect on gene expression through the variation of activity of regulatory elements, such as promoters and enhancers. In particular, we identified cases of promoters compensation with no evidence of differences in the total gene expression. In the second part of the thesis we investigated the cellular variability of gene expression in two different contexts: firstly, we studied the X chromosome inactivation (XCI) happening in female individuals. We observe extensive heterogeneity of this mechanism within and between individuals and tissues. And secondly, we integrated single-cell gene expression quantification and DNA variation to identify cell-type specific expression quantitative trait loci (eQTLs) in the human pancreatic islets

Chemical modulators of fibrinogen production and their impact on venous thrombosis

Thrombosis is a leading cause of morbidity and mortality. Fibrinogen, the soluble substrate for fibrin-based clotting, has a central role in haemostasis and thrombosis and its plasma concentration correlates with cardiovascular disease event risk and a prothrombotic state in experimental models. We aimed to identify chemical entities capable of changing fibrinogen production and test their impact on experimental thrombosis. A total of 1,280 bioactive compounds were screened for their ability to alter fibrinogen production by hepatocyte-derived cancer cells and a selected panel was tested in zebrafish larvae. Anthralin and all-trans retinoic acid (RA) were identified as fibrinogen-lowering and fibrinogen-increasing moieties, respectively. In zebrafish larvae, anthralin prolonged laser-induced venous- occlusion times and reduced thrombocyte accumulation at injury sites. RA had opposite effects. Treatment with RA, a nuclear receptor ligand, increased fibrinogen mRNA levels. Using an antisense morpholino oligonucleotide to deplete zebrafish fibrinogen, we correlated a shortening of laser-induced venous thrombosis times with RA treatment and fibrinogen protein levels. Anthralin had little effect on fibrinogen mRNA in zebrafish larvae, despite leading to lower detectable fibrinogen. Therefore, we made a proteomic scan of anthralin-treated cells and larvae. A reduced representation of proteins linked to the canonical secretory pathway was detected, suggesting that anthralin affects protein secretion. In summary, we found that chemical modulation of fibrinogen levels correlates with measured effects on experimental venous thrombosis and could be investigated as a therapeutic avenue for thrombosis prevention

18F-FDG PET/MRI for Rectal Cancer TNM Restaging After Preoperative Chemoradiotherapy: Initial Experience

OBJECTIVE: F-18-FDG-PET/MRI is a novel hybrid techinque that has been recently introduced in oncological imaging, showing promising results. The aim of this study is to assess the value of whole-body F-18-FDG-PET/MRI for predicting the pathological stage of locally advanced rectal cancer after preoperative chemoradiotherapy. DESIGN: This was a prospective observational study. SETTINGS: The study was conducted at a tertiary care hospital. PATIENTS: Thirty-six patients with locally advanced rectal cancer (25 male, median age 68.5 years) were prospectively assessed with PET/MRI and thoracoabdominal CT before and after preoperative chemoradiotherapy. Twenty-seven patients underwent low anterior or abdominoperineal resection. Nine patients with a complete clinical response underwent organ-preserving treatment (8 local excision and 1 watch-and-wait approach) with > 1-year follow-up. MAIN OUTCOME MEASURES: One radiologist evaluated pelvic MRI and CT. A second radiologist and a nuclear medicine physician jointly assessed PET/MRI. The imaging was compared with histology or follow-up (ypT0 vs T >= 1 and ypN0 vs ypN+ categories). Metastases were confirmed with biopsy or a follow-up CT scan at least at 1 year after preoperative chemoradiotherapy. The sensitivity, specificity, and accuracy values of the imaging techniques were calculated using standard formulas. RESULTS: The accuracy for ypT staging was 89% and 92%, and the accuracy for ypN was 86% and 92% for MRI and PET/MRI. Compared with CT, PET/MRI correctly diagnosed 4 of 5 metastases, but it did not detect a lung metastatic nodule. In 11% of the patients, the PET/MRI changed the treatment strategy. LIMITATIONS: This study is limited by its small sample size. CONCLUSIONS: Although the whole-body PET/MRI was more accurate than the pelvic MRI alone for the prediction of tumor and node response to preoperative chemoradiotherapy, the technique performed worse than CT in detecting small lung metastasis

MBV: a method to solve sample mislabeling and detect technical bias in large combined genotype and sequencing assay datasets

Abstract Motivation Large genomic datasets combining genotype and sequence data, such as for expression quantitative trait loci (eQTL) detection, require perfect matching between both data types. Results We described here MBV (Match BAM to VCF); a method to quickly solve sample mislabeling and detect cross-sample contamination and PCR amplification bias. Availability and Implementation MBV is implemented in C ++ as an independent component of the QTLtools software package, the binary and source codes are freely available at https://qtltools.github.io/qtltools/. Supplementary information Supplementary data are available at Bioinformatics online

Extrachromosomal driver mutations in glioblastoma and low-grade glioma

Alteration of the number of copies of double minutes (DMs) with oncogenic EGFR mutations in response to tyrosine kinase inhibitors is a novel adaptive mechanism of glioblastoma. Here we provide evidence that such mutations in DMs, called here amplification-linked extrachromosomal mutations (ALEMs), originate extrachromosomally and could therefore be completely eliminated from the cancer cells. By exome sequencing of seven glioblastoma patients we reveal ALEMs in EGFR, PDGFRA and other genes. These mutations together with DMs are lost by cancer cells in culture. We confirm the extrachromosomal origin of such mutations by showing that wild-type and mutated DMs may coexist in the same tumour. Analysis of 4,198 tumours suggests the presence of ALEMs across different tumour types with the highest prevalence in glioblastomas and low-grade gliomas. The extrachromosomal nature of ALEMs explains the observed drastic changes in the amounts of mutated oncogenes (like EGFR or PDGFRA) in glioblastoma in response to environmental changes

The Mediator complex colocalizes with the SIM2 DNA binding sites.

<p>Frequency distribution of MED1 (<b>a</b>) and MED12 (<b>b</b>) DNA binding sites in a 40kb window centered to the SIM2 peaks. Pie charts show the proportion of SIM2 DNA binding sites overlapping MED1 or MED12 DNA binding sites (in grey) (100bp window). p = Fisher’s exact test p-value; F score: measure of the significance of the association (1 = perfect match).</p

Identification and characterization of the SIM2 DNA binding sites by ChIP-seq.

<p><b>a.</b> Venn diagram of the number of SIM2 binding sites identified by ChIP-seq in each SIM2 clones (A6, B8, C4) and EB3 line. The sum of the bold numbers is equal to the 1229 SIM2 DNA binding sites found in at least 2 SIM2 clones. <b>b.</b> The pie chart shows the genomic distribution of these 1229 sites. <b>c.</b> Distribution of the distances between the SIM2 DNA binding sites and the closest transcription start site (TSS). <b>d.</b> Selection of gene ontology terms significantly over-represented in the list of genes associated to a SIM2 DNA binding site.</p

List of Sim2 target genes identified using the ChIA-PET data.

<p>List of Sim2 target genes identified using the ChIA-PET data.</p